Heptamethine cyanine dyes have attracted considerable interest because of their ability to fluoresce in the near infrared (NIR) region. However, the utility of such dyes in bioassays and in vivo applications has been limited because many dyes of this class exhibit photo-instability, poor water solubility, and a relatively small Stokes shift, typically less than about 25 nm. The Stokes shift is the difference (in wavelength or frequency units) between the peak absorption and emission spectra for the same electronic transition. Dyes having a small Stoke shifts frequently lack sensitivity due to self-quenching and interference by excitation and scattered light.
Recently, there have been reports of heptamethine cyanine dyes that exhibit increased Stokes shift propoerties. For example, Pham et al. described the preparation and properties of a near infrared dye, 4-sulfonir, having a large Stokes shift. See Pham et al., Chem. Commun. (2008), 1895-97. Other enamine-containing dyes have been disclosed by Peng et al. to have a large Stokes shift and strong fluorescence. See Peng et al., J. Am. Chem. Soc. (2005), 127:4170-71. Related dyes and dye conjugates have been reported by Wang et al. as diagnostic contrast agents. See Wang et al., U.S. Publication No. 20080206886 (published Aug. 28, 2008).
In spite of these advances, there remains a need to identify heptamethine cyanine dyes that are both photostable and chemically stable, and have an increased Stokes shifts, as well as a high quantum yield and molar extinction coefficient. Dyes exhibiting good water solubility suitable for use in bioassays are particularly desirable.